LETTERS TO THE EDITOR 983
J ALLERGY CLIN IMMUNOL VOLUME 134, NUMBER 4
From athe Immunology, Allergy and Rheumatology Unit, Division of Pediatrics, bthe Millennium Institute on Immunology and Immunotherapy, and cthe Department of Rheumatology and Clinical Immunology at the School of Medicine, Pontificia Universidad Cat olica de Chile, Santiago, Chile; and dthe Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, NC. E-mail: [email protected]. Disclosure of potential conflicts of interest: A. Borzutzky has been a speaker for Roche and Novartis Laboratories (Chile); has received research support from Fondo Nacional de Desarrollo Cientıfico y Tecnologico (FONDECYT grant nos. 1130615 and 1130387) (Chile) and FONIS (grant no. SA13I20173) (Chile); has received lecture fees from Roche Laboratories (Chile) and Pfizer (Chile); and has received travel support from Pfizer (Chile) and Abbott Laboratories (Chile). V. Mezzano has received lecture fees from Brystol-Myers Squibb and has received support for travel, accommodations, and meeting registration from Janssen. A. W. Burks is or has been on the board for Food Allergy Research and Education, the American Academy of Allergy, Asthma & Immunology, and the Food Allergy Initiative; has received consultancy fees from Dynavax Technologies Corp, Genalyte, GLG Research, Perrigo Company, Regeneron Pharmaceuticals, Perosphere Inc, Dow AgroSciences, ExploraMed Development, McNeil Nutritionals, Merck, Nordic Biotech, Norvartis Pharma AG, Nutricia North American, Portola Pharaceuticals, Publicis Healthcare Communications Group, and Unilever; has received research support from Hycor Biomedical; has received lecture fees from Abbott Laboratories, the American College of Allergy, Asthma, and Immunology, and Mylan Speciality; has received royalties from UpToDate; has received payment for development of educational presentations from Levine Children’s Hospital and Oak Ridge Associated Universities; and has stock/stock options in Allertein and MastCell Pharmaceuticals. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Greenberger PA. Idiopathic anaphylaxis. Immunol Allergy Clin North Am 2007; 27:273-93, vii-viii. 2. Lieberman P, Nicklas RA, Oppenheimer J, Kemp SF, Lang DM, Bernstein DI, et al. The diagnosis and management of anaphylaxis practice parameter: 2010 update. J Allergy Clin Immunol 2010;126:477-80, e1-42. 3. Ditto AM, Harris KE, Krasnick J, Miller MA, Patterson R. Idiopathic anaphylaxis: a series of 335 cases. Ann Allergy Asthma Immunol 1996;77:285-91. 4. Tejedor Alonso MA, Sastre DJ, Sanchez-Hernandez JJ, Perez FC, de la Hoz Caballer B. Idiopathic anaphylaxis: a descriptive study of 81 patients in Spain. Ann Allergy Asthma Immunol 2002;88:313-8. 5. Grammer LC, Shaughnessy MA, Harris KE, Goolsby CL. Lymphocyte subsets and activation markers in patients with acute episodes of idiopathic anaphylaxis. Ann Allergy Asthma Immunol 2000;85:368-71. 6. Warrier P, Casale TB. Omalizumab in idiopathic anaphylaxis. Ann Allergy Asthma Immunol 2009;102:257-8. 7. Arkwright PD. Anti-CD20 or anti-IgE therapy for severe chronic autoimmune urticaria. J Allergy Clin Immunol 2009;123:510-1; author reply 1. 8. Chakravarty SD, Yee AF, Paget SA. Rituximab successfully treats refractory chronic autoimmune urticaria caused by IgE receptor autoantibodies. J Allergy Clin Immunol 2011;128:1354-5. 9. Mallipeddi R, Grattan CE. Lack of response of severe steroid-dependent chronic urticaria to rituximab. Clin Exp Dermatol 2007;32:333-4. 10. Gamerman S, Singh AM, Makhija M, Sharathkumar A. Successful eradication of inhibitor in a patient with severe haemophilia B and anaphylaxis to factor IX concentrates: is there a role for Rituximab and desensitization therapy? Haemophilia 2013;19:e382-5. Available online July 10, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.032
Lessons in gene hunting: A RAG1 mutation presenting with agammaglobulinemia and absence of B cells To the Editor: Recombination activating genes (RAG) 1 and 2 are critical for Variable (V), Diversity (D) and Joining (J) (V[D]J) recombination and lymphocyte development.1 Mutations in RAG1/2 associated with less than 1% of wild-type recombination activity typically result in severe combined immunodeficiency (SCID) lacking T and B cells.2 Hypomorphic mutations with residual RAG activity
can result in Omenn syndrome, characterized by erythroderma, lymphadenopathy, and the presence of oligoclonal T cells. More recently, studies have broadened the phenotypic variability associated with RAG1/2 mutations, which now includes granulomatous disease, gd T-cell expansion, CD41 lymphopenia with intact T-cell function, and hyper-IgM syndrome.1,3 We report a patient with a homozygous missense mutation in RAG1, who presented initially with agammaglobulinemia, absent B cells, and normal numbers of T cells, followed by evolution into a T2B2 natural killer (NK1) SCID phenotype with opportunistic infections and granulomas. The patient was born to Kuwaiti first-degree cousin parents and presented at age 6 months with pneumonia and failure to thrive. The physical examination was notable for the absence of tonsils. Computed tomography of the chest revealed a prominent thymus. He had undetectable levels of IgG and IgA. The IgM level was 30 mg/dL. The absolute lymphocyte count was 5010 cells/mL, with normal numbers of total CD31 cells, CD41 and CD81 T cells, CD161/CD561 NK cells, but virtually no CD191 B cells (Table I). Genetic testing did not reveal any mutations in the genes encoding Bruton tyrosine kinase, m heavy chain, Iga, Igb, l5, and VpreB. He was given a diagnosis of agammaglobulinemia with absent B cells and was treated with intravenous immunoglobulin. The patient subsequently developed recurrent otitis media and cytomegalovirus-associated retinitis at age 4 years. Immunologic evaluation performed at age 5 years revealed virtually absent B cells, CD31 and CD41 T-cell lymphopenia, and normal numbers of NK cells (Table I). Lymphocyte proliferation to PHA was severely decreased (Table I). NK cell cytotoxic function was normal (data not shown). At age 8 years, he developed fever and diarrhea because of EBV viremia and Salmonella enteritidis. At age 10 years, the patient developed persistent colitis; endoscopy and colonoscopy revealed esophageal candidiasis and colonic granulomas. At that time, he had progressive T-cell lymphopenia with a predominantly CD45RO1 activated phenotype, and nearly absent CD41CD45RA1CD311 recent thymic emigrants (Table I). HLA typing of the patient and his mother revealed no evidence of maternal engraftment. T-cell proliferation to PHA and anti-CD3 stimulation was minimal. Proliferation to anti-CD3 stimulation increased with the addition of anti-CD28, suggesting residual signaling through the CD3 pathway. The combination of absent B cells, agammaglobulinemia, and progressive T-cell lymphopenia with poor T-cell function led to a diagnosis of combined immunodeficiency. The patient died at the age of 12 years from sepsis. Whole genome sequencing was performed on DNA obtained from the patient before his death, his father, and his healthy sibling. Thirteen candidate genes had novel mutations that were homozygous in the patient, heterozygous in the father, and heterozygous or absent in the sibling (see Table E1 in this article’s Online Repository at www.jacionline.org). Only the c.1211G>A mutation in RAG1 correlated with the patient’s phenotype of combined immunodeficiency. This mutation resulted in the substitution of arginine with glutamine at position 404 (p.R404Q) in the nonamer-binding region of the protein. The nonamerbinding region of RAG1 binds to recombination signal sequences that flank the V(D)J elements of the immunoglobulin and T-cell receptor genes, and is critical for V(D)J recombination activity.7 Using a flow cytometry–based assay that permits analysis of RAG1 recombination activity on an intrachromosomal substrate,8 we have demonstrated that the R404Q RAG1 mutant protein is
984 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL OCTOBER 2014
TABLE I. Immune profiles Proband Laboratory values
6 mo 4
Lymphocytes (cells/mL) (normal range) CD31 CD31CD41 CD31CD81 CD41/CD81 ratio CD41CD45RA1 CD41CD45RO1 CD41CD45RA1CD311 CD191 CD161/CD561 Eosinophils (cells/mL) (normal range)5 Immunoglobulins (mg/dL) (normal range)6 IgG IgA IgM Proliferation (cpm) (normal control)* Phytohemagglutinin Anti-CD3 Anti-CD31CD28 Background
5y
11 y
1,550 1,150 388 679 0.51
5,010 (3,400-9,000) 4,520 (1,900-5,900) 3,300 (1,400-4,300) 1,220 (500-1,700) 2.7 (1.6-3.8) ND ND ND 2 (610-2,600) 340 (160-950) 184 (200-300)
1,620 541 260 279 0.93 3.7 95 0.96 61 1,004 210
(2,300-5,400) (1,400-3,700) (700-2,200) (490-1,300) (0.9-2.6) ND ND ND 4 (390-1,400) 349 (130-720) 95 (0-600)
Undetectable (215-704) Undetectable (8.1-68) 30 (35-102)
ND ND ND
ND ND ND
1,578 (16,485) ND ND 32 (192)
ND ND ND ND
(1,900-3,700) (1,200-2,600) (650-1,500) (370-1,100) (0.9-3.4) (46%-77%) (13%-30%) (45.7%)* (270-860) (100-480) (0-600)
541 232 6797 131
(65,369) (3,814) (19,170) (221.5)
ND, Not determined. Values in boldface and italics are abnormal. *Values in parentheses for lymphocyte proliferation are derived from studies done on a healthy control the same day.
104 10
R404Q 94.1
0.323
10
3
102
hCD2
104
101 5.56 100 100 101
1.98e 102
103
-3
104
WT 69.7
23.5
104 10
3
102
101
101 0.309 102
103
104
0
3
102
100 6.55 100 101
Mock 96
4.02 100 100 101
0 102
103
104
GFP FIG 1. Recombination activity of the RAG1 R404Q mutant protein. Abelson (Abl) virus-transformed Rag12/2 mouse pro-B cells engineered to contain a single integrant of an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences (RSS) were transduced with retroviral vectors allowing expression of wild-type (WT) human RAG1 or the mutant R404Q RAG1 protein, and human CD2 (hCD2) as a reporter. Mock-transduced cells served as a negative control. After MACS-purification of hCD2-expressing cells and treatment in vitro with imatinib, recombination activity of the R404Q mutant protein was measured by normalizing GFP expression to levels observed in the presence of WT RAG1, as previously described.8 One representative experiment of 3 is shown. MACS, Magnetic cell sorting.
normally expressed, but has markedly reduced recombination activity (1.18% 6 0.14% of wild-type) (Fig 1). This mutation has been previously reported in patients with T2B2 SCID or Omenn syndrome.9 In contrast to previously published reports of patients with this mutation, our patient presented with clinical and laboratory features of agammaglobulinemia with absent B cells, a prominent thymus, normal numbers of circulating T cells, and no features of Omenn syndrome such as erythroderma or eosinophilia. Normal numbers of T cells with absence of B cells have been associated with another RAG1 mutation (c.T2686C, p.W896A) in a patient who presented with isolated culturenegative pneumonitis at age 6 months.10 This patient, like ours, had a normal thymic shadow and normal T-cell numbers. The W896A mutation resulted in impaired proliferation to mitogens and a highly restricted TCRb repertoire that was not of maternal origin. We have previously demonstrated that the V(D)J
recombination activity of the W896A mutant is associated with minimally preserved RAG1 function (0.9% 6 0.1% of wildtype),8 similar to the R404Q mutation in our patient. This case broadens the spectrum of disease associated with a RAG1 mutation that nearly abolishes recombination activity. This phenomenon has been seen in families in which the same mutation gives rise to different clinical and immunological phenotypes, suggesting that the manifestations of RAG1/2 mutations may reflect interactions with other uncharacterized genetic modifiers, epigenetic factors, or environmental exposures. The progression of the patient’s disease also underscores how a single mutation can have variable time-dependent effects in the same individual. Although he had normal numbers of T cells at age 6 months, T-cell function may have been abnormal at that time as suggested by his history of pneumonia and failure to thrive. He developed progressively worsening T-cell numbers and function,
LETTERS TO THE EDITOR 985
J ALLERGY CLIN IMMUNOL VOLUME 134, NUMBER 4
opportunistic infections, granulomas, and ultimately died of sepsis, which, in retrospect, are consistent with an underlying RAG1/2 mutation. The diagnostic delay in this case underscores the importance of SCID newborn screening, particularly in areas of the world where lymphocyte proliferation or T-cell receptor repertoire studies are not available. Quantification of T-cell receptor excision circles can identify poor thymic output, even in the case of normal T-cell numbers due to maternal engraftment or oligoclonality.11 In addition, cases of SCID that present with absent B-cell numbers, as in our patient and others,10 support the inclusion of quantification of k-deleting recombination excision circles, which can detect absent B cells, as part of newborn screening for SCID for improved early detection.12 Mona Hedayat, MDa* Michel J. Massaad, PhDa* Yu Nee Lee, PhDa* Mary Ellen Conley, MDb Jordan S. Orange, MD, PhDc Toshiro K. Ohsumi, PhDd Waleed Al-Herz, MDe Luigi D. Notarangelo, MDaà Raif S. Geha, MDaà Janet Chou, MDaà From athe Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Mass; bSt Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY; cImmunology, Allergy, and Rheumatology, Baylor College of Medicine and the Texas Children’s Hospital, Houston, Tex; dthe Department of Molecular Biology, Massachusetts General Hospital, Boston, Mass; and ethe Faculty of Medicine, Department of Pediatrics, Kuwait University, Kuwait City, Kuwait. E-mail: [email protected]. *These authors contributed equally to this work as first authors. àThese authors contributed equally to this work as last authors. This study was supported by the National Institutes of Health (grant no. NIH P01AI076210 and grant no. AI-094017), the Dubai Harvard Foundation for Medical Research (to R.S.G.), a Manton Foundation Pilot Award (to J.C.), and the Kuwait Foundation for the Advancement of Sciences (grant no. 2010-1302-05 to W.A.-H.). Disclosure of potential conflict of interest: W. Al-Herz has received research support from the Kuwait Foundation for the Advancement of Sciences. R. S. Geha has received research support from the National Institutes of Health (grant nos. NIH P01 AI-076210 and AI-094017) and the Dubai Harvard Foundation for Medical Research. J. Chou has received research support from the Manton Foundation. The rest of the authors declare that they have no relevant conflicts of interest. REFERENCES 1. Niehues T, Perez-Becker R, Schuetz C. More than just SCID–the phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2. Clin Immunol 2010;135:183-92. 2. Notarangelo LD, Villa A, Schwarz K. RAG and RAG defects. Curr Opin Immunol 1999;11:435-42. 3. Chou J, Hanna-Wakim R, Tirosh I, Kane J, Fraulino D, Lee YN, et al. A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome. J Allergy Clin Immunol 2012;130:1414-6. 4. Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm ER, et al. Pediatric AIDS Clinical Trial Group. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003;112:973-80. 5. Tietz NW, editor. Clinical guide to laboratory tests (3rd ed.). Philadelphia, Pa: W.B. Saunders; 1995. 6. Jolliff CR, Cost KM, Stivrins PC, Grossman PP, Nolte CR, Franco SM, et al. Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry. Clin Chem 1982;28:126-8. 7. Kirch SA, Sudarsanam P, Oettinger MA. Regions of RAG1 protein critical for V(D)J recombination. Eur J Immunol 1996;26:886-91. 8. Lee Y, Frugoni F, Dobbs K, Walter JE, Giliani S, Gennery AR, et al. A systematic analysis of recombination activity and genotype-phenotype correlation in human RAG1-deficiency. J Allergy Clin Immunol 2014;133:1099-108. 9. Sobacchi C, Marrella V, Rucci F, Vezzoni P, Villa A. RAG-dependent primary immunodeficiencies. Hum Mutat 2006;27:1174-84.
10. Zhang J, Quintal L, Atkinson A, Williams B, Grunebaum E, Roifman CM. Novel RAG1 mutation in a case of severe combined immunodeficiency. Pediatrics 2005; 116:e445-9. 11. Puck JM. Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: the winner is T-cell receptor excision circles. J Allergy Clin Immunol 2012;129:607-16. 12. Borte S, von Dobeln U, Fasth A, Wang N, Janzi M, Winiarski J, et al. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR. Blood 2012;119:2552-5. Available online June 27, 2014. http://dx.doi.org/10.1016/j.jaci.2014.04.037
IgE allergen component-based profiling and atopic manifestations in patients with Netherton syndrome To the Editor: Netherton syndrome (NS; OMIM no. 266500) is an autosomal recessive genodermatosis with atopic dermatitis (AD)–like erythroderma, a bamboo-like hair-shaft defect, and multiple atopic manifestations: asthma, urticaria, angioedema, allergic rhinitis, increased IgE levels, and blood hypereosinophilia.1 NS is caused by serine protease inhibitor, Kazal type 5 (SPINK5) mutations leading to defective lymphoepithelial Kazal-type related inhibitor (LEKTI), a serine protease inhibitor expressed in the upper epidermal skin layers.1 Loss of LEKTI inhibition results in a severe skin barrier defect.1 SPINK5 polymorphisms, especially the 420Lys LEKTI variant, have been associated with atopy and AD.2 We studied in detail the atopic symptoms in 10 Finnish patients with NS with SPINK5 mutations. Our patients with NS had multiple allergies initiating neonatally and increasing rapidly in early childhood (Table I and see Tables E1 and E2 in this article’s Online Repository at www.jacionline.org). Increased serum total IgE levels (up to 17,433 kU/L) were seen in 9 of 10 patients, and 8 of 10 had blood hypereosinophilia (Table I and see Table E2). Urticaria, asthma, and angioedema were common (Table I). We evaluated the IgE antibody profile specifically with the ImmunoCAP ISAC microarray (Thermo Fisher Scientific, Waltham, Mass) in 6 patients with NS 0.4 to 49.5 years of age, enabling the most precise IgE profiling in patients with NS to date (see the Methods section in this article’s Online Repository at www.jacionline.org). Repeated ISAC microarray testing within the first 3 years for 2 patients (patients T4 and T6) showed that the initial IgE antibodies increased in intensity and new IgE sensitizations occurred rapidly for both, reaching 17% to 21% at 2 years and 35% by 3.2 years of age (Fig 1 and see Table E1). A similar trend in increasing specific IgE (sIgE) levels to multiple allergens with increasing age was noted for other patients (see Table E2). Overall, the sIgE antibodies to allergens triggering clinical symptoms represented common food allergens, such as egg, cow’s milk, wheat, and nuts, as well as pollens and animal proteins (see Table E1). All patients reacted against 14% to 37% of the allergen components. In comparison, considering that our study was not a case-control study, a cohort of 140 German pediatric patients with AD studied on an ISAC microarray with 95 allergen components showed mean sensitization to 7.6 6 7.7 of allergens, ranging from 0% (in 14% of patients) to 34%, and higher numbers of sIgE responses associated with higher total IgE levels (3-26,567 kU/L).3 All our patients with NS had IgE antibodies to multiple potentially anaphylactic stable allergens, such as lipid transfer proteins (LTPs; 83%), storage proteins (17% to 33%), and tropomyosin (33%; see Table E1).4
985.e1 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL OCTOBER 2014
TABLE E1. Candidate genes* identified by whole genome sequencing Gene
BSDC1 OSCP1 MPHOSPH8 CERK FAM203B C9orf57 RAG1 EPPK1 OR52A5 ZNF284 SPAG8 PHF2 LCAP
Full name
BSD domain containing 1 Organic solute carrier partner 1 M-phase phosphoprotein 8 Ceramide kinase Family with sequence similarity 203, member B Chromosome 9 open reading frame 57 Recombination activating gene Epiplakin 1 Olfactory receptor, family 52, subfamily A, member 5 Zinc finger protein 284 Sperm-associated antigen 8 PHD finger protein 2 Lung carcinoma–associated protein 10
*Candidate genes are defined as genes with novel mutations in coding regions or canonical splice sites that were homozygous in the patient, heterozygous in his father, and either heterozygous or absent in his healthy brother. Novel mutations are defined as those absent from public polymorphism databases (dbSNP and 1000 Genomes) and our in-house database of 73 Middle Eastern exomes/genomes.
J ALLERGY CLIN IMMUNOL VOLUME 134, NUMBER 4
From athe Immunology, Allergy and Rheumatology Unit, Division of Pediatrics, bthe Millennium Institute on Immunology and Immunotherapy, and cthe Department of Rheumatology and Clinical Immunology at the School of Medicine, Pontificia Universidad Cat olica de Chile, Santiago, Chile; and dthe Department of Pediatrics, University of North Carolina School of Medicine, Chapel Hill, NC. E-mail: [email protected]. Disclosure of potential conflicts of interest: A. Borzutzky has been a speaker for Roche and Novartis Laboratories (Chile); has received research support from Fondo Nacional de Desarrollo Cientıfico y Tecnologico (FONDECYT grant nos. 1130615 and 1130387) (Chile) and FONIS (grant no. SA13I20173) (Chile); has received lecture fees from Roche Laboratories (Chile) and Pfizer (Chile); and has received travel support from Pfizer (Chile) and Abbott Laboratories (Chile). V. Mezzano has received lecture fees from Brystol-Myers Squibb and has received support for travel, accommodations, and meeting registration from Janssen. A. W. Burks is or has been on the board for Food Allergy Research and Education, the American Academy of Allergy, Asthma & Immunology, and the Food Allergy Initiative; has received consultancy fees from Dynavax Technologies Corp, Genalyte, GLG Research, Perrigo Company, Regeneron Pharmaceuticals, Perosphere Inc, Dow AgroSciences, ExploraMed Development, McNeil Nutritionals, Merck, Nordic Biotech, Norvartis Pharma AG, Nutricia North American, Portola Pharaceuticals, Publicis Healthcare Communications Group, and Unilever; has received research support from Hycor Biomedical; has received lecture fees from Abbott Laboratories, the American College of Allergy, Asthma, and Immunology, and Mylan Speciality; has received royalties from UpToDate; has received payment for development of educational presentations from Levine Children’s Hospital and Oak Ridge Associated Universities; and has stock/stock options in Allertein and MastCell Pharmaceuticals. The rest of the authors declare that they have no relevant conflicts of interest.
REFERENCES 1. Greenberger PA. Idiopathic anaphylaxis. Immunol Allergy Clin North Am 2007; 27:273-93, vii-viii. 2. Lieberman P, Nicklas RA, Oppenheimer J, Kemp SF, Lang DM, Bernstein DI, et al. The diagnosis and management of anaphylaxis practice parameter: 2010 update. J Allergy Clin Immunol 2010;126:477-80, e1-42. 3. Ditto AM, Harris KE, Krasnick J, Miller MA, Patterson R. Idiopathic anaphylaxis: a series of 335 cases. Ann Allergy Asthma Immunol 1996;77:285-91. 4. Tejedor Alonso MA, Sastre DJ, Sanchez-Hernandez JJ, Perez FC, de la Hoz Caballer B. Idiopathic anaphylaxis: a descriptive study of 81 patients in Spain. Ann Allergy Asthma Immunol 2002;88:313-8. 5. Grammer LC, Shaughnessy MA, Harris KE, Goolsby CL. Lymphocyte subsets and activation markers in patients with acute episodes of idiopathic anaphylaxis. Ann Allergy Asthma Immunol 2000;85:368-71. 6. Warrier P, Casale TB. Omalizumab in idiopathic anaphylaxis. Ann Allergy Asthma Immunol 2009;102:257-8. 7. Arkwright PD. Anti-CD20 or anti-IgE therapy for severe chronic autoimmune urticaria. J Allergy Clin Immunol 2009;123:510-1; author reply 1. 8. Chakravarty SD, Yee AF, Paget SA. Rituximab successfully treats refractory chronic autoimmune urticaria caused by IgE receptor autoantibodies. J Allergy Clin Immunol 2011;128:1354-5. 9. Mallipeddi R, Grattan CE. Lack of response of severe steroid-dependent chronic urticaria to rituximab. Clin Exp Dermatol 2007;32:333-4. 10. Gamerman S, Singh AM, Makhija M, Sharathkumar A. Successful eradication of inhibitor in a patient with severe haemophilia B and anaphylaxis to factor IX concentrates: is there a role for Rituximab and desensitization therapy? Haemophilia 2013;19:e382-5. Available online July 10, 2014. http://dx.doi.org/10.1016/j.jaci.2014.05.032
Lessons in gene hunting: A RAG1 mutation presenting with agammaglobulinemia and absence of B cells To the Editor: Recombination activating genes (RAG) 1 and 2 are critical for Variable (V), Diversity (D) and Joining (J) (V[D]J) recombination and lymphocyte development.1 Mutations in RAG1/2 associated with less than 1% of wild-type recombination activity typically result in severe combined immunodeficiency (SCID) lacking T and B cells.2 Hypomorphic mutations with residual RAG activity
can result in Omenn syndrome, characterized by erythroderma, lymphadenopathy, and the presence of oligoclonal T cells. More recently, studies have broadened the phenotypic variability associated with RAG1/2 mutations, which now includes granulomatous disease, gd T-cell expansion, CD41 lymphopenia with intact T-cell function, and hyper-IgM syndrome.1,3 We report a patient with a homozygous missense mutation in RAG1, who presented initially with agammaglobulinemia, absent B cells, and normal numbers of T cells, followed by evolution into a T2B2 natural killer (NK1) SCID phenotype with opportunistic infections and granulomas. The patient was born to Kuwaiti first-degree cousin parents and presented at age 6 months with pneumonia and failure to thrive. The physical examination was notable for the absence of tonsils. Computed tomography of the chest revealed a prominent thymus. He had undetectable levels of IgG and IgA. The IgM level was 30 mg/dL. The absolute lymphocyte count was 5010 cells/mL, with normal numbers of total CD31 cells, CD41 and CD81 T cells, CD161/CD561 NK cells, but virtually no CD191 B cells (Table I). Genetic testing did not reveal any mutations in the genes encoding Bruton tyrosine kinase, m heavy chain, Iga, Igb, l5, and VpreB. He was given a diagnosis of agammaglobulinemia with absent B cells and was treated with intravenous immunoglobulin. The patient subsequently developed recurrent otitis media and cytomegalovirus-associated retinitis at age 4 years. Immunologic evaluation performed at age 5 years revealed virtually absent B cells, CD31 and CD41 T-cell lymphopenia, and normal numbers of NK cells (Table I). Lymphocyte proliferation to PHA was severely decreased (Table I). NK cell cytotoxic function was normal (data not shown). At age 8 years, he developed fever and diarrhea because of EBV viremia and Salmonella enteritidis. At age 10 years, the patient developed persistent colitis; endoscopy and colonoscopy revealed esophageal candidiasis and colonic granulomas. At that time, he had progressive T-cell lymphopenia with a predominantly CD45RO1 activated phenotype, and nearly absent CD41CD45RA1CD311 recent thymic emigrants (Table I). HLA typing of the patient and his mother revealed no evidence of maternal engraftment. T-cell proliferation to PHA and anti-CD3 stimulation was minimal. Proliferation to anti-CD3 stimulation increased with the addition of anti-CD28, suggesting residual signaling through the CD3 pathway. The combination of absent B cells, agammaglobulinemia, and progressive T-cell lymphopenia with poor T-cell function led to a diagnosis of combined immunodeficiency. The patient died at the age of 12 years from sepsis. Whole genome sequencing was performed on DNA obtained from the patient before his death, his father, and his healthy sibling. Thirteen candidate genes had novel mutations that were homozygous in the patient, heterozygous in the father, and heterozygous or absent in the sibling (see Table E1 in this article’s Online Repository at www.jacionline.org). Only the c.1211G>A mutation in RAG1 correlated with the patient’s phenotype of combined immunodeficiency. This mutation resulted in the substitution of arginine with glutamine at position 404 (p.R404Q) in the nonamer-binding region of the protein. The nonamerbinding region of RAG1 binds to recombination signal sequences that flank the V(D)J elements of the immunoglobulin and T-cell receptor genes, and is critical for V(D)J recombination activity.7 Using a flow cytometry–based assay that permits analysis of RAG1 recombination activity on an intrachromosomal substrate,8 we have demonstrated that the R404Q RAG1 mutant protein is
984 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL OCTOBER 2014
TABLE I. Immune profiles Proband Laboratory values
6 mo 4
Lymphocytes (cells/mL) (normal range) CD31 CD31CD41 CD31CD81 CD41/CD81 ratio CD41CD45RA1 CD41CD45RO1 CD41CD45RA1CD311 CD191 CD161/CD561 Eosinophils (cells/mL) (normal range)5 Immunoglobulins (mg/dL) (normal range)6 IgG IgA IgM Proliferation (cpm) (normal control)* Phytohemagglutinin Anti-CD3 Anti-CD31CD28 Background
5y
11 y
1,550 1,150 388 679 0.51
5,010 (3,400-9,000) 4,520 (1,900-5,900) 3,300 (1,400-4,300) 1,220 (500-1,700) 2.7 (1.6-3.8) ND ND ND 2 (610-2,600) 340 (160-950) 184 (200-300)
1,620 541 260 279 0.93 3.7 95 0.96 61 1,004 210
(2,300-5,400) (1,400-3,700) (700-2,200) (490-1,300) (0.9-2.6) ND ND ND 4 (390-1,400) 349 (130-720) 95 (0-600)
Undetectable (215-704) Undetectable (8.1-68) 30 (35-102)
ND ND ND
ND ND ND
1,578 (16,485) ND ND 32 (192)
ND ND ND ND
(1,900-3,700) (1,200-2,600) (650-1,500) (370-1,100) (0.9-3.4) (46%-77%) (13%-30%) (45.7%)* (270-860) (100-480) (0-600)
541 232 6797 131
(65,369) (3,814) (19,170) (221.5)
ND, Not determined. Values in boldface and italics are abnormal. *Values in parentheses for lymphocyte proliferation are derived from studies done on a healthy control the same day.
104 10
R404Q 94.1
0.323
10
3
102
hCD2
104
101 5.56 100 100 101
1.98e 102
103
-3
104
WT 69.7
23.5
104 10
3
102
101
101 0.309 102
103
104
0
3
102
100 6.55 100 101
Mock 96
4.02 100 100 101
0 102
103
104
GFP FIG 1. Recombination activity of the RAG1 R404Q mutant protein. Abelson (Abl) virus-transformed Rag12/2 mouse pro-B cells engineered to contain a single integrant of an inverted green fluorescent protein (GFP) cassette flanked by recombination signal sequences (RSS) were transduced with retroviral vectors allowing expression of wild-type (WT) human RAG1 or the mutant R404Q RAG1 protein, and human CD2 (hCD2) as a reporter. Mock-transduced cells served as a negative control. After MACS-purification of hCD2-expressing cells and treatment in vitro with imatinib, recombination activity of the R404Q mutant protein was measured by normalizing GFP expression to levels observed in the presence of WT RAG1, as previously described.8 One representative experiment of 3 is shown. MACS, Magnetic cell sorting.
normally expressed, but has markedly reduced recombination activity (1.18% 6 0.14% of wild-type) (Fig 1). This mutation has been previously reported in patients with T2B2 SCID or Omenn syndrome.9 In contrast to previously published reports of patients with this mutation, our patient presented with clinical and laboratory features of agammaglobulinemia with absent B cells, a prominent thymus, normal numbers of circulating T cells, and no features of Omenn syndrome such as erythroderma or eosinophilia. Normal numbers of T cells with absence of B cells have been associated with another RAG1 mutation (c.T2686C, p.W896A) in a patient who presented with isolated culturenegative pneumonitis at age 6 months.10 This patient, like ours, had a normal thymic shadow and normal T-cell numbers. The W896A mutation resulted in impaired proliferation to mitogens and a highly restricted TCRb repertoire that was not of maternal origin. We have previously demonstrated that the V(D)J
recombination activity of the W896A mutant is associated with minimally preserved RAG1 function (0.9% 6 0.1% of wildtype),8 similar to the R404Q mutation in our patient. This case broadens the spectrum of disease associated with a RAG1 mutation that nearly abolishes recombination activity. This phenomenon has been seen in families in which the same mutation gives rise to different clinical and immunological phenotypes, suggesting that the manifestations of RAG1/2 mutations may reflect interactions with other uncharacterized genetic modifiers, epigenetic factors, or environmental exposures. The progression of the patient’s disease also underscores how a single mutation can have variable time-dependent effects in the same individual. Although he had normal numbers of T cells at age 6 months, T-cell function may have been abnormal at that time as suggested by his history of pneumonia and failure to thrive. He developed progressively worsening T-cell numbers and function,
LETTERS TO THE EDITOR 985
J ALLERGY CLIN IMMUNOL VOLUME 134, NUMBER 4
opportunistic infections, granulomas, and ultimately died of sepsis, which, in retrospect, are consistent with an underlying RAG1/2 mutation. The diagnostic delay in this case underscores the importance of SCID newborn screening, particularly in areas of the world where lymphocyte proliferation or T-cell receptor repertoire studies are not available. Quantification of T-cell receptor excision circles can identify poor thymic output, even in the case of normal T-cell numbers due to maternal engraftment or oligoclonality.11 In addition, cases of SCID that present with absent B-cell numbers, as in our patient and others,10 support the inclusion of quantification of k-deleting recombination excision circles, which can detect absent B cells, as part of newborn screening for SCID for improved early detection.12 Mona Hedayat, MDa* Michel J. Massaad, PhDa* Yu Nee Lee, PhDa* Mary Ellen Conley, MDb Jordan S. Orange, MD, PhDc Toshiro K. Ohsumi, PhDd Waleed Al-Herz, MDe Luigi D. Notarangelo, MDaà Raif S. Geha, MDaà Janet Chou, MDaà From athe Division of Immunology, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Mass; bSt Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, NY; cImmunology, Allergy, and Rheumatology, Baylor College of Medicine and the Texas Children’s Hospital, Houston, Tex; dthe Department of Molecular Biology, Massachusetts General Hospital, Boston, Mass; and ethe Faculty of Medicine, Department of Pediatrics, Kuwait University, Kuwait City, Kuwait. E-mail: [email protected]. *These authors contributed equally to this work as first authors. àThese authors contributed equally to this work as last authors. This study was supported by the National Institutes of Health (grant no. NIH P01AI076210 and grant no. AI-094017), the Dubai Harvard Foundation for Medical Research (to R.S.G.), a Manton Foundation Pilot Award (to J.C.), and the Kuwait Foundation for the Advancement of Sciences (grant no. 2010-1302-05 to W.A.-H.). Disclosure of potential conflict of interest: W. Al-Herz has received research support from the Kuwait Foundation for the Advancement of Sciences. R. S. Geha has received research support from the National Institutes of Health (grant nos. NIH P01 AI-076210 and AI-094017) and the Dubai Harvard Foundation for Medical Research. J. Chou has received research support from the Manton Foundation. The rest of the authors declare that they have no relevant conflicts of interest. REFERENCES 1. Niehues T, Perez-Becker R, Schuetz C. More than just SCID–the phenotypic range of combined immunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2. Clin Immunol 2010;135:183-92. 2. Notarangelo LD, Villa A, Schwarz K. RAG and RAG defects. Curr Opin Immunol 1999;11:435-42. 3. Chou J, Hanna-Wakim R, Tirosh I, Kane J, Fraulino D, Lee YN, et al. A novel homozygous mutation in recombination activating gene 2 in 2 relatives with different clinical phenotypes: Omenn syndrome and hyper-IgM syndrome. J Allergy Clin Immunol 2012;130:1414-6. 4. Shearer WT, Rosenblatt HM, Gelman RS, Oyomopito R, Plaeger S, Stiehm ER, et al. Pediatric AIDS Clinical Trial Group. Lymphocyte subsets in healthy children from birth through 18 years of age: the Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin Immunol 2003;112:973-80. 5. Tietz NW, editor. Clinical guide to laboratory tests (3rd ed.). Philadelphia, Pa: W.B. Saunders; 1995. 6. Jolliff CR, Cost KM, Stivrins PC, Grossman PP, Nolte CR, Franco SM, et al. Reference intervals for serum IgG, IgA, IgM, C3, and C4 as determined by rate nephelometry. Clin Chem 1982;28:126-8. 7. Kirch SA, Sudarsanam P, Oettinger MA. Regions of RAG1 protein critical for V(D)J recombination. Eur J Immunol 1996;26:886-91. 8. Lee Y, Frugoni F, Dobbs K, Walter JE, Giliani S, Gennery AR, et al. A systematic analysis of recombination activity and genotype-phenotype correlation in human RAG1-deficiency. J Allergy Clin Immunol 2014;133:1099-108. 9. Sobacchi C, Marrella V, Rucci F, Vezzoni P, Villa A. RAG-dependent primary immunodeficiencies. Hum Mutat 2006;27:1174-84.
10. Zhang J, Quintal L, Atkinson A, Williams B, Grunebaum E, Roifman CM. Novel RAG1 mutation in a case of severe combined immunodeficiency. Pediatrics 2005; 116:e445-9. 11. Puck JM. Laboratory technology for population-based screening for severe combined immunodeficiency in neonates: the winner is T-cell receptor excision circles. J Allergy Clin Immunol 2012;129:607-16. 12. Borte S, von Dobeln U, Fasth A, Wang N, Janzi M, Winiarski J, et al. Neonatal screening for severe primary immunodeficiency diseases using high-throughput triplex real-time PCR. Blood 2012;119:2552-5. Available online June 27, 2014. http://dx.doi.org/10.1016/j.jaci.2014.04.037
IgE allergen component-based profiling and atopic manifestations in patients with Netherton syndrome To the Editor: Netherton syndrome (NS; OMIM no. 266500) is an autosomal recessive genodermatosis with atopic dermatitis (AD)–like erythroderma, a bamboo-like hair-shaft defect, and multiple atopic manifestations: asthma, urticaria, angioedema, allergic rhinitis, increased IgE levels, and blood hypereosinophilia.1 NS is caused by serine protease inhibitor, Kazal type 5 (SPINK5) mutations leading to defective lymphoepithelial Kazal-type related inhibitor (LEKTI), a serine protease inhibitor expressed in the upper epidermal skin layers.1 Loss of LEKTI inhibition results in a severe skin barrier defect.1 SPINK5 polymorphisms, especially the 420Lys LEKTI variant, have been associated with atopy and AD.2 We studied in detail the atopic symptoms in 10 Finnish patients with NS with SPINK5 mutations. Our patients with NS had multiple allergies initiating neonatally and increasing rapidly in early childhood (Table I and see Tables E1 and E2 in this article’s Online Repository at www.jacionline.org). Increased serum total IgE levels (up to 17,433 kU/L) were seen in 9 of 10 patients, and 8 of 10 had blood hypereosinophilia (Table I and see Table E2). Urticaria, asthma, and angioedema were common (Table I). We evaluated the IgE antibody profile specifically with the ImmunoCAP ISAC microarray (Thermo Fisher Scientific, Waltham, Mass) in 6 patients with NS 0.4 to 49.5 years of age, enabling the most precise IgE profiling in patients with NS to date (see the Methods section in this article’s Online Repository at www.jacionline.org). Repeated ISAC microarray testing within the first 3 years for 2 patients (patients T4 and T6) showed that the initial IgE antibodies increased in intensity and new IgE sensitizations occurred rapidly for both, reaching 17% to 21% at 2 years and 35% by 3.2 years of age (Fig 1 and see Table E1). A similar trend in increasing specific IgE (sIgE) levels to multiple allergens with increasing age was noted for other patients (see Table E2). Overall, the sIgE antibodies to allergens triggering clinical symptoms represented common food allergens, such as egg, cow’s milk, wheat, and nuts, as well as pollens and animal proteins (see Table E1). All patients reacted against 14% to 37% of the allergen components. In comparison, considering that our study was not a case-control study, a cohort of 140 German pediatric patients with AD studied on an ISAC microarray with 95 allergen components showed mean sensitization to 7.6 6 7.7 of allergens, ranging from 0% (in 14% of patients) to 34%, and higher numbers of sIgE responses associated with higher total IgE levels (3-26,567 kU/L).3 All our patients with NS had IgE antibodies to multiple potentially anaphylactic stable allergens, such as lipid transfer proteins (LTPs; 83%), storage proteins (17% to 33%), and tropomyosin (33%; see Table E1).4
985.e1 LETTERS TO THE EDITOR
J ALLERGY CLIN IMMUNOL OCTOBER 2014
TABLE E1. Candidate genes* identified by whole genome sequencing Gene
BSDC1 OSCP1 MPHOSPH8 CERK FAM203B C9orf57 RAG1 EPPK1 OR52A5 ZNF284 SPAG8 PHF2 LCAP
Full name
BSD domain containing 1 Organic solute carrier partner 1 M-phase phosphoprotein 8 Ceramide kinase Family with sequence similarity 203, member B Chromosome 9 open reading frame 57 Recombination activating gene Epiplakin 1 Olfactory receptor, family 52, subfamily A, member 5 Zinc finger protein 284 Sperm-associated antigen 8 PHD finger protein 2 Lung carcinoma–associated protein 10
*Candidate genes are defined as genes with novel mutations in coding regions or canonical splice sites that were homozygous in the patient, heterozygous in his father, and either heterozygous or absent in his healthy brother. Novel mutations are defined as those absent from public polymorphism databases (dbSNP and 1000 Genomes) and our in-house database of 73 Middle Eastern exomes/genomes.
